Tetrazolium salts, such as 2-(4-iodophenyl)-3- (4-nitrophenyl)-5-phenyl tetrazolium (INT), are very useful in the measurement of analytes which can be converted to an equivalent concentration of NADH due to the reduction of the tetrazolium salt to its corresponding formazan which reduction can be accurately measured by colorimetric means.
A typical reagent system for determining glucose concentration in body fluids is based on reductive chemistry wherein the primary components are hexokinase (HK), adenosine triphosphate (ATP), glucose-6-phosphate dehydrogenase (G-6-PDH), diaphorase, nicotinamideadenine dinucleotide (AND) and a tetrazolium salt as indicator. In operation, hexokinase catalyzes the reaction in which, in the presence of glucose, a phosphate radical is taken from ATP thereby converting it to adenosine diphosphate to form glucose-6-phosphate which is oxidized in the presence of AND and G-6-PDH thereby reducing AND to NADH. The NADH, in the presence of diaphorase as electron acceptor, reduces the colorless tetrazolium salt to its colored formazan counterpart thereby providing a detectable response. The reaction steps, as represented by the following scheme, represent the determination of NADH as an indirect means of determining the glucose concentration in the test sample: ##STR1##
The utility of tetrazolium salts in such systems for detecting such analytes is proportional to their solubility in water or suitable organic solvents. This is particularly true in the case of dry reagent diagnostic test devices, such as those in which a tetrazolium salt is dissolved in a polar organic solvent for impregnation into a carrier matrix such as paper or a polymer matrix or dissolved in an aqueous solution of a film forming polymer such as gelatin. Tetrazolium salt indicators are typically used with gelatin film and other dry reagent formulations which employ diaphorase or a chemical mediator in the color generating step. An adequate amount of indicator must be present to completely consume the reducing equivalents that originate from the influx of an analyte such as glucose. In most cases, in order to obtain a reasonably thin coating of the film forming polymer and to provide a sufficient supply of the indicator within the porous matrix, the concentration of indicator must be in the range of 0.05M to 0.15M or more.
U.S. Pat. No. 1,892,019 discloses the increased water solubility of benzylmorphine after it is reacted with alkyl sulphonic acid, e.g. methane or ethanesulfonic acid, by formation of the corresponding salt. U.S. Pat. No. 4,334,071 discloses the enhancement of the solubility of 17-cyclobutylmethyl-3-hydroxy-862 -methyl-6-methylene morphinane by converting its chloride salt to the corresponding methanesulfonate.
U.S. Pat. No. 3,655,382 discloses tetrazolium thiazolium salts in which the counteranion can be chloride, iodide, bromide, thiocyanate, thiosulfate, sulfate, paratoluenesulfonate, methylsulfate, ethyl sulfate, nitrate, acetate, perchlorate, perborate, sulfite, hydroxide or carbonate.
In U.S. Pat. No. 4,221,864 the patentees state that one of the objects of their invention is to provide a novel light sensitive photographic material containing a tetrazolium compound. They point out that this and other objects can be attained by preparing a photographic material which comprises a support and at least one light sensitive silver halide layer and another hydrophylic colloidal layer coated on the support, one of which layer contains a tetrazolium salt. They point out that where the salt of a tetrazolium compound is used as a non-diffusible ingredient, such a salt can be synthesized by reacting a tetrazolium cation with an anion capable of making the selected compound non-diffusible. Counteranions such as those derived from higher alkylbenzenesulfonic acids, e.g. dodecylbenzenesulfonic acid or a higher alkyl sulfuric acid ester such as lauryl sulfate are disclosed.